Citation of a reference herein shall not be construed as an admission that such reference is prior art to the present invention.
2.1. Fucosyltransferases
Fucosyltransferases are enzymes that catalyze the addition of a fucose residue to a terminal galactose acceptor of saccharide precursors. Fucosyltransferase activity is involved in the production of oligosaccharides, glycolipids or glycoproteins. There are four known classes of fucosyltransferases, namely those that catalyze the addition of fucose in α1→2, α1→3, α1→4 and α1→6 linkages.
Fucosyltransferases are best known for their roles in the synthesis of the oligosaccharide moieties that comprise blood group antigenic determinants. For example, the fucosyltransferase encoded by the H gene catalyzes the transfer of fucose in an α1→2 linkage to the terminal galactose of Gal(β1-4)GlcNAc(β1-3)Gal-R to produce ‘H substance’ on the surface of erythrocytes. Further addition of N-acetylgalactosamine or galactose leads to the formation of the type A or type B blood group substances respectively. An analogous enzyme encoded by the Se locus catalyzes the formation of ‘H substance’ in epithelial tissues for secretion rather than presentation at the cell surface (Rosen et al., 1989, Dictionary of Immunology, Stockton Press, New York, pp. 1-3).
Previous experiments with H35 hepatoma cell extracts demonstrated that transfer of fucose to neolacto-series acceptors occurred at a rate only 2% of that found for GM1 (Holmes, E. H., et al, 1983, J. Biol. Chem, 258:3706-3713). This substrate specificity is more restricted compared to other cloned α1→2fucosyltransferases but is most closely related to secretor-type enzymes (Larsen, R. D., et al., 1990, Proc. Natl. Acad. Sci. USA 87:6674-6678; Kelly, R. J., et al., 1995, J. Biol. Chem. 270:4640-4649; Hitoshi, S., et al., 1995, J. Biol. Chem. 270:8844-8850; Hitoshi, S., et al., 1996, J. Biol. Chem. 271:16975-16981).
2.2. Structure of α1→2Fucosyltransferases
To date, a number of genes encoding H-type and Se-type α1→2fucosyltransferases have been cloned from several species of organisms. Three human α1→2fucosyltransferases (Larsen et al., 1990, Biochemistry 87:6674-6678; Koda et al., 1997, Eur. J. Biochem. 246:750-755; Kelly et al., 1995, J. Biol. Chem. 270:4640-4649), three rabbit α1→2fucosyltransferases (known as RFT-I, RFT-II and RFT-III) (Hitoshi et al., 30 1995, J. Biol. Chem. 270:8844-8850; Hitoshi et al., 1996, J. Biol. Chem. 271:16975-19681), and two mouse α1→2fucosyltransferases (Tsuji, 1996, GenBank accession no. Y09882; Lin et al., 1998, GenBank accession no. AF064792) have been described. Piau et al. (1994, Eur. J. Biochem. 300:623-626) disclose fragments, designated FTA and FTB, of two rat α1→2fucosyltransferases isolated from rat PROb colon adenocarcinoma cells. Piau et al. showed that antisense expression of the FTA or FTB nucleic acid fragments inhibited the endogenous α1→2fucosyltransferase activity of PROb cells with respect to the synthetic fucose acceptor phenyl β-D-galactopyranoside; however the FTB fragment was not shown to be sufficient for α1→2fucosyltransferase catalytic activity, nor was the substrate specificity of the PROb α1→2fucosyltransferase activity determined.
H-type α1→2fucosyltransferases are membrane localized whereas Se-type α1→2fucosyltransferases are localized to the Golgi apparatus. Amino acid sequence alignment of membrane bound H-type α1→2fucosyltransferases reveals that, like other glycosyltransferases, there exists a homologous domain structure comprising a short intracellular N-terminal domain, a transmembrane domain, an extracellular stem region not required for enzymatic activity, and finally, the catalytic domain at the C-terminus. Generally, there is little sequence homology outside the catalytic domain.
2.3. Ganglioside GM1 and its Fucosylated Derivative Fucosyl-GM1 
Gangliosides are cell surface constituents comprising glycosphingolipids (produced by the linking of ceramides to oligosaccharides) with sialic acid residues. Depending on the number of sialic acid residues they possess, gangliosides are known as mono-, di-, tri- or polysialogangliosides. GM1 stands for ganglioside mono(sialic acid)1.
Fucosyl-GM1, detected by monoclonal antibodies, is found largely in the nervous system, and in particular on a subpopulation of neurons in the dorsal root ganglia and dorsal horn of the spinal cord, as well as on surrounding satellite cells surrounding the fucosyl-GM1 positive neurons (Kusunoki et al., 1989, Brain Res. 494:391-395; Kusonoki et al., 1992, Neurosci. Res. 15:74-80).
Gangliosides have long been implicated in diseased states. They are often prominent cell surface constituents of transformed cells (see Section 2.5, infra) and alterations in their metabolism give rise to diseases of the nervous system. For example, several fatal hereditary diseases are caused by lysosomal storage of gangliosides wherein the absence or deficiency of lysosomal enzymes results in the deleterious accumulation of gangliosides. The most well known of these diseases is the neurodegenerative Tay-Sachs disease, which is characterized by the accumulation of ganglioside GM2. Accumulation of GM1 results in GM1 Gangliosidosis.
2.4. Regulation of Fucosyltransferase Expression
‘H substance’, the fucosylated precursor of blood group determinants, is strictly regulated temporally and spatially during vertebrate development (Fenderson et al., 1986, Dev. Biol. 114:12-21).
Dramatic changes in the expression of cell surface glycolipids are found with oncogenesis (Hakomori, 1989, Adv. Cancer Res. 52:257-331; Alhadeff, 1989, CRC Crit. Rev. Oncol./Hematol. 9:37-107). These changes frequently are oncofetal in nature in that a particular carbohydrate structure may be expressed during normal fetal development, disappear in adult tissues, and reappear in association with oncogenesis giving rise to a premalignant or malignant marker. One such example is expression of the ganglio-B determinant (II3NeuAcIV3 αGalIV2FucGg4) during early stages of chemical carcinogenesis in rat liver with N-2-acetylaminofluorene (AAF) (Holmes and Hakomori, 1982, J. Biol. Chem. 257:7698-7703; Scribner et al., 1983, Environ. Health Perspect. 49:81-89). Expression of this determinant has been shown to be a property of liver parenchymal cells resulting from a carcinogenic stimulus but not hepatotoxicity (Holmes, 1990, Carcinogenesis 11:89-94). This determinant has also been shown to be developmentally regulated in rat stomach (Bonhours et al., 1987, J. Biol. Chem. 258:3706-3713). Expression of this antigen is due to the activation of an α1→2fucosyltransferase which is normally unexpressed in adult rat liver parenchymal cells. This enzyme efficiently transfers fucose onto the terminal galactose residue of a GM1 precursor, producing fucosyl-GM1 (IV3NeuAcIV2FucGgOse4Cer). Fucosyl-GM1 is a substrate for a constituitively expressed α1→3galactosyltransferase forming the blood group B determinant on a ganglioside core chain (Holmes and Hakomori, 1983, J. Biol. Chem. 258:3706-3713; Holmes and Hakomori, 1987, J. Biochem. 258:3706-3713). This α1→3galactosyltransferase behaves as a blood group B transferase in that it efficiently catalyzes transfer of galactose in α1→3-linkage to terminal galactose residues of α1→2fucosylated neolacto- and ganglio-series acceptors (Holmes and Hakomori, 1983, J. Biol. Chem. 258:3706-3713).
High α1→2fucosyltransferase expression is observed in rat hepatoma H35 cells (Holmes and Hakomori, 1983, J. Biol. Chem. 258:3706-3713; Holmes and Hakomori, 1987, J. Biochem. 258:3706-3713). The enzyme from H35 cells has specificity for a ganglio-series core chain. These cells accumulate large amounts of fucosyl-GM, (Baumann, H., et al., 1979, Cancer Res. 39:2637-2643). Enzymological studies indicated this enzyme was inhibited by a wide variety of detergents, an unusual property for a membrane bound glycosyltransferase (Holmes, E. H., et al, 1983, J. Biol. Chem, 258:3706-3713). This property may reflect a role for membrane phospholipids in maintaining the enzyme in an active conformation (Holmes and Hakomori, 1987, J. Biochem. 101: 1095-1105). Later studies demonstrated that active enzyme could be solubilized from H35 cell membranes by 0.4% CHAPSO which bound to the affinity resin GDP-hexanolamine-Sepharose (Holmes, E. H., et al., 1987, J. Biochem. 101:1095-1105).
Further, the observation about the production by transformed cells of high levels of fucosyl-GM1 as a result of α1→2fucosyltransferase activity, is not restricted to rat hepatoma cells. For example, in humans, fucosyl-GM1 is associated with small cell lung carcinoma (Fredman et al., 1986, Biochim. Biophys. Acta 875:316-323; Nilsson et al., 1984, Glycoconjugate J. 1:43-49).
Generally, enzymatic oligosaccharide synthesis (including synthesis of glycolipids, glycoproteins, etc.) has been limited by the difficulty of isolation and enrichment of glycosyltransferases from natural sources. Thus, there is a need for methods to produce easily isolatable quantities of glycosyltranferases with high enzymatic activity. Such glycosyltransferases, produced, e.g. in vitro, would be useful reagents in compensating for the lack of natural resources. In particular, there is a need for methods to produce easily isolatable GM1-specific α1→2fucosyltransferase. The ability to synthesize fucosyl-GM1 in vitro is of particularly high value, as the ganglioside is important for the development of the mammalian nervous system. GM1-specific α1→2fucosyltransferase can be used to catalyze the addition of fucose residues to terminal Galβ1→3 GalNAc saccharide chains of glycoproteins, glycolipids, glycolipoproteins and oligosaccharides, producing saccharide compositions that are useful nutritional additives or bases therefor. Further, fucosyl-GM1 is envisaged to be an important tool in cancer therapy and cancer diagnostics. Until the cloning and characterization of the nucleic acid and amino acid sequences of the catalytic domain and the full length α1→2fucosyltransferase of the present invention, no α1→2fucosyltransferases with GM1 specificity had been identified.